1. Technical Field
The present invention relates to the field of nucleic acids and in particular to detection methods relating thereto. It further relates to the diagnosis of various infectious diseases by such detection. It further relates to the synthesis of amplified biotin-labelled DNA sequences by polymerase chain reaction techniques, and the detection of such sequences by microtiter plate capture. It still further relates to novel primers and capture probes that are capable of detecting Chlamydia trachomatis.
2. Description of Related Disclosures
U.S. Pat. Nos. 4,683,195 and 4,683,202 (both incorporated herein by reference) disclose methods of amplifying DNA sequences by a technique now known in the art as "polymerase chain reaction" (PCR). The polymerase chain reaction is a procedure in which DNA is specifically amplified by multiple primer extension syntheses of complementary strands (Saiki et al, Science, 230: 1350-1354 and 239: 489-491; 1985, 1988). The PCR product, amplified up to 10.sup.6 -10.sup.7 fold, is a DNA fragment of discrete size (amplicon) which can be detected by gel electrophoresis, or by other means as described herein. Briefly, PCR involves the preparation of short oligonucleotide primers which correspond to opposing ends of a known "target" sequence which one intends to amplify and subsequently detect. In this Procedure, DNA or RNA is extracted from cells, tissues, body fluids and the like. The nucleic acid is denatured and the oligonucleotide primers are added in molar excess, along with dNTPs (deoxyribonucleotide triphosphates) and a DNA polymerase enzyme, such as preferably heat stable Tao polymerase. Upon subsequent heat denaturing, cooling to allow annealing to primers, and primer extension by DNA Polymerase, two "long products", which begin with the respective primers, are produced, complementary to the two original strands. This procedure is repeated, and after a second cycle two original strands, two long products from cycle 1, two new "long products", and two "short products" are produced. The length of these short products (amplicons) is equal to the number of nucleotides between and including both primers. With additional cycles, additional "long products" are produced, increasing in a linear fashion with each cycle. However, the generation of amplicons increases at an exponential rate with each cycle, and by means of this amplification, the detection of extremely small quantities of DNA is enabled.
Through the use of PCR technology, the detection of specific DNA sequences present in minute quantities is possible. Several of these techniques involve the use of various hybridized probes affixed to certain materials.
As will be fully described later, the present invention utilizes fixation of a capture DNA sequence to microtiter wells and subsequent detection by hybridization to labelled (viz. biotinylated) amplicons. It also involves the use of guanidine thiocyanate in the hybridization step. Several procedures have been described in the literature which are relevant herein. Two such reports describe the immobilization of large target DNAs to microtiter wells, followed by the hybridization of these targets to biotinylated oligonucleotide or nick-translated DNA probes.
Cook et al., Nucleic Acids Research. 16:4077-4095 (1988) immobilized bacteriophage M13 target DNA on microtiter wells of Immulon.sup. 2 plates by incubating target DNA in 1 M ammonium acetate for 1.5-2 hours at 37.degree. C. The immobilized target DNA was detected after hybridization to terminal-biotin labelled or internal-biotin labelled oligonucleotide probes, followed by addition of streptavidin-HRP complex and hydrogen peroxide/o-phenylene diamine (OPD) for colorimetric detection of hybridization.
Nagata et al., FEBS Lett., 183:379-382 (1985) immobilized lambda target DNA in microtiter wells in PBS containing 0.1 M MgCl.sub.2. After an overnight room temperature incubation followed by removal of the solution, the plate was irradiated with ultraviolet light. Hybridization with a biotinylated (nick-translated) lambda DNA probe was followed by complexing with avidin-betagalactosidase. Fluorescence was measured after the addition of the substrate, 4-methylumbelliferyl-beta-D-galactoside.
Hevey et al., U.S. Pat. No. 4,228,237, described a method which uses biotin reacting to avidin (covalently attached to an enzyme) to detect a ligand in liquid medium. Our present method differs in that the ligand is directly labelled with biotin, not an intermediate biotin labelled anti-ligand. Further, various other prior disclosures describe specific hybridization probes and diagnostic uses thereof; e.g., U.S. Pat. No. 4,358,535 issued to Falkow and EP 63,879 to Ward. The latter describes the use of biotin-labelled DNA probes detected by enzymes linked to avidin or biotin-specific antibodies.
Ranki et al., U.S. Pat. No. 4,486,539, describes the use of two non-overlapping nucleic acid reagents (one bound to a solid carrier and the other being labelled) to detect and identify microbes by DNA hybridization. The present invention differs in that the target nucleic acid is itself labelled, and does not require the use of an additional labelled nucleic acid probe for detection.
Stabinsky, U.S. Pat. No. 4,751,177, described a DNA detection method wherein the target DNA is hybridized in solution to a mediator polynucleotide and to a labelled probe polynucleotide. The mediator polynucleotide, in turn, is complementary to a polynucleotide immobilized on a solid support. The present invention differs in that (a) the target is labelled during amplification; no labelled reporter group is needed for detection, and (b) the capture probe is bound directly to a solid support, without the use of a mediator polynucleotide.
Guanadine thiocyanate (GuSCN) has been used in cell extraction and subsequent nucleic acid hybridization. For example, Thompson and Gillespie, Anal. Biochem., 163:281-291 (1987), prepared radiolabelled RNA probes to detect target DNA or RNA. In a dot blot format, the .sup.32 P-labelled probe was hybridized in 5 M GuSCN/0.1 M ethylenediaminetetraacetic acid (EDTA), disodium salt, pH 8.0. Pellegrino et al., Biotechniques, 452-459 (1987), described a solution hybridization to detect HIV-RNA in blood cells. Blood cells were dissolved in 5 M GuSCN/0.1 M EDTA and hybridized with a radiolabelled RNA probe in the same solution at room temperature. TCA-precipitated hybrids were collected on membranes and radioactivity was determined by scintillation counting. Gillespie, International Patent Application #PCT/US87/01023, (1987), described the use of guanidine thiocyanate for molecular hybridization using a labelled probe to detect target DNA bound to a solid support. The patent describes the use of GuSCN for molecular hybridization over a concentration range of 3 M-6.5 M at ambient temperature. The format for such hybridization includes binding target nucleic acid to nitrocellulose or nylon membrane (dot blot or Southern blot), prehybridization, hybridization to a radiolabelled Probe in GuSCN, washing and detection by autoradiography.